Quantitative phosphoproteomics studies reveal different signaling cascades in cultured distal convoluted tubular cells upon vasopressin or angiotensin II stimulation (1109.10)

Abstract

The thiazide‐sensitive NaCl cotransporter (NCC) plays an important role in sodium reabsorption in the kidney distal convoluted tubules (DCT). Both angiotensin II (ANGII) and vasopressin (AVP) are hormones known to exert effects in this segment and induce phosphorylation of NCC, which regulates NCC function. However, the signaling pathway from AVP to NCC and possible interplay between AVP and ANGII signaling cascades remain unknown. Here we performed large scale quantitative phosphoproteomics of cultured mpkDCT cells to map global changes in protein phosphorylation events upon treatment with ANG II or the V2‐selective analog dDAVP (30min). Experiments were performed using the stable isotope labeling by amino acids in cell culture (SILAC) quantification approach. LC‐ESI‐MS/MS identified 21616 quantifiable phosphopeptides and 12848 different phosphorylation sites, of which 7149 were confidently assigned . Kinase prediction of the regulated phosphorylation sites using NetworKIN indicated that ANGII stimulation increased activity of the kinase families CDK, MAPKs or PAK, whereas dDAVP stimulation increased the activity of the kinases PKA, DMPK, PIM2, PAK, or CLK. Motif analysis indicated that RRXSXXX (PKA substrate motif) was a preferential target for upregulated phosphorylation sites upon dDAVP stimulation, whereas for downregulated phosphorylation sites XXXSPXX (CDK substrate motif) was more prominent. cAMP assays showed that dDAVP induced an increase in cAMP levels in mpkDCT cells, supporting the PKA signaling cascades identified from proteomic analysis. Our results suggest that highly distinct signaling cascades are activated in cultured mpkDCT cells upon acute treatment by either dDAVP or ANG II, analysis of which will likely identify novel mechanisms for NCC regulation.Grant Funding Source: Supported by Lundbeck Foundation and the Danish Medical Research Foudation